The appearance of vancomycin-resistant strains among clinical isolates of methicillin-resistant (and multiresistant) Staphylococcus aureus places a special urgency on efforts to better understand the mechanism of beta-lactam and glycopeptide resistance in these pathogens. The Research Program will have four main foci. (A) A major new development, namely, the successful "activation" of the silent mecA homologue of Staphylococcus sciuri and its introduction into S. aureus, will be followed up by a combination of genetic and biochemical experiments. (B) and (C): New data indicate that the mechanisms of resistance to both methicillin and vancomycin involve a complex stress response in which numerous genetic determinants participate and which is induced by addition of the antibiotic to the growth medium. The drastic alteration in cell wall composition that accompanies expression of resistance will be followed by high-resolution biochemical techniques. The complete genome of the highly and homogeneously methicillin-resistant S. aureus (MRSA) strain COL is currently being sequenced at The Institute for Genomic Research (TIGR). With the genome sequence in hand, the plan is to construct a microarray system to explore gene networks operating in the expression of resistance to methicillin and vancomycin. The microarrays will be used to initially define a global transcription profile of COL. This profile will then serve as a basis for comparison in experiments designed to identify changes in gene expression that occur upon the addition of antibiotics or under the influence of specific mutations affecting resistance. Part (D) will concentrate on attempts to identify unique genetic determinants of vancomycin resistance in mutants of COL and in clinical isolates by a combination of transposon mutagenesis and the microarray technology. The project should provide new insights into the mechanisms of beta-lactam and glycopeptide resistance, hitherto unknown genetic and biochemical features of staphylococcal cell wall metabolism, and it may also contribute to the identification of new antibacterial targets.